The research proposed here is designed to identify subpopulations of lung fibroblasts which differ in their responsiveness to various growth promoting factors and in their sensitivity to signals which stimulate the production or secretion of collagen. Regulation of cell growth and protein synthesis in response to extracellular signals commonly involves the generation of intracellular second messengers, among which Ca++ is one of the most important. The recent development of fluorescent dyes which change their spectral properties on binding Ca++ now makes it possible to accurately measure levels of free Ca++ in individual cells. When combined with digital image processing, this technology allows visualization of subtle changes in the subcellular distribution of free Ca++. This technique is ideally suited to the identification of subpopulations of fibroblasts since individual cells which differ in their responses to extracellular signals can be identified and cloned on the same culture dish. We will apply this new technology to the classification of lung fibroblasts into categories based on their responses to a variety of growth factors. In conjunction with these studies on cell proliferation, we will also characterize the lung fibroblast population for heterogeneity in the synthesis and secretion of collagen. Antibodies to types I and III collagen will be used in conjunction with digital image analysis to quantitate the amount of collagen produced by individual cells, while in situ hybridization will be used to measure the cellular content of mRNA coding for these collagen subtypes. In these ways, we will test the hypothesis that there are, among the several classes of fibroblasts present in the normal lung, one or more subclasses which posses the combined properties of being especially sensitive to growth stimulation and active in the synthesis of collagen. Initially, all studies will be conducted on primary or very early passage cultures of rat lung fibroblasts. Populations of fibroblasts derived from animals induced to develop pulmonary fibrosis by exposure to drugs or environmental irritants to the lung will be analyzed and compared to populations of normal lung fibroblasts in order to determine whether there is an enrichment of particular subtypes in the fibrotic lung. In subsequent studies, clonal lines representing discrete fibroblast subclasses will be established and tested for the degree to which they are maintaining their growth and collagen synthesizing characteristics.